In cell mediated immunity, a disease causing agent, such as a virus, is engulfed by a specialized cell called the antigen presenting cell (hereinafter "APC"). The APC breaks up the virus and fragments the antigenic determinants of the virus, i.e., viral specific polypeptides, into polypeptide fragments. These fragmented antigenic determinants are then transported to the cell surface of the APC. At this time, the APC also produces or modifies the major histocompatability complex molecules Class I and Class II (hereinafter "MHC Class I" or "MHC Class II", respectively), which are heavily involved in cell mediated immunity and which are produced within and transported to the surface of the APC. MHC Class I molecules specifically bind to cytotoxic/suppressor T cells (Tc/s) and MHC Class II molecules specifically bind to helper/accessory T cells (Th). The MHC molecules contain at least two binding sites, i.e., an antigen binding site known as agretope binding site, which is highly variable between MHC molecules for different agretopes, and a site which binds to the T cell, i.e., the T cell specific binding ligand, which is highly conserved (see Bjorkman, P. J. et al, Nature, 329:506 (1987) and Bjorkman, P. J. et al, Nature, 329:512 (1987)).
T cells are activated by the combination of (1) the binding of the fragmented antigenic determinants, present on the surface of the APC, to the surface of the T cells and (2) the binding of the highly conserved region of the MHC molecules, present on the surface of the APC, to the surface of the T cells. Usually, binding by the fragmented antigenic determinants or the highly conserved region of the MHC molecules alone to the surface of the T cells does not give rise to activation of the T cells since to do so would give rise to an unregulated and indiscriminate polyclonal activation of most, if not all, T cells and could result in pathogenic conditions. The binding of the MHC molecules to the surface of the T cells is in part mediated through the agretope, i.e., the binding of the antigen to the MHC molecules acts as a signal to the MHC molecules to bind to the surface of the T cells (see Bjorkman, P. J., Nature, 229:506 (1987)). In addition, the MHC molecule contains recognition sites so that unless the APC and the T cell contain the same NHC molecules with the same genetic composition, they are recognized as "not-self" and the desired interaction cannot successfully occur. The resulting activated T cells can then recognize the disease causing or associated agent, e.g., virus infected cell, tumor cell, etc., in the bloodstream or elsewhere, as foreign and acts to kill such. This gives rise to cell mediated immunity to the disease caused by, e.g., the virus, without any antibody, or humoral immunity, involvement.
The APC, typically a macrophage, also produces and releases Interleukin 1 (hereinafter "IL-1") as a consequence of the interaction and processing of the antigen. IL-1 interacts with the T cell as a part of the activation process of the T cell. IL-1 causes activated T cells to produce Interleukin-2 (hereinafter "IL-2"). However, as with most hormones, IL-1 activity is generalized, i.e., it is not specific to a particular antigen but, rather, is involved in invoking a generalized inflammatory response.
Since MHC molecules are very large and highly polymorphic, antigenicity problems arise when administering such to a subject. Further, there is a high variability of agretopes and MHC molecules. Thus, it is difficult to isolate an appropriate MHC molecule for a disease causing or associated agent of interest so as to be able to form a complex thereof which can thereby activate T cells specific to a disease of interest.
In an embodiment of the present invention, the above-discussed problem is overcome by employing only a portion of the MHC molecules which bind to T cells, i.e., the highly conserved region thereof, and covalently linking such to an antigen associated with disease or causative agent of disease, or epitope thereof of interest, thereby forming a heterofunctional cellular immunological reagent and avoiding the necessity of isolating suitable or using large and polymorphic, MHC molecules. Further, in some cases, one of the reasons for a failure to respond to an antigen is a lack of antigen processing and/or appropriate MHC molecules. The heterofunctional cellular immunological reagent of the present invention overcomes this problem.
The clinical and industrial immunologists working in AIDS have not focused on the correlation of cell mediated immunity and disease since most of their assays are based on humoral immunity mechanisms. Cell mediated immunity, because of its slow reactions, the requirement for a living cell derived from an intact host and the MHC restriction inherent in the system have deflected attention away from cell mediated immunity. Cell mediated immunity has remained, therefore, somewhat of a "black box" with inputs and outputs defined but little understood in the way of internal mechanisms.
The rapidity of the re-activation of AIDS or herpes viruses indicates that re-activation cannot be a result of the breakdown of humoral response mechanisms. That is, re-activation occurs in a short number of days while serum antibodies are still abundant. In part because of the above circumstantial evidence in humoral response and in part because of other evidence in cellular mechanisms, a breakdown of cell mediated response mechanisms is implicated in re-activation of these disease causing agents.
In the case of tuberculosis (hereinafter "TB"), another disease where cellular immunity is paramount, IL-2, also known as T cell growth factor, can restore the in vitro cellular immune response to the mycobacterium. Since IL-1 is available in subjects afflicted with TB, a defect in the stimulation of IL-2 production by the cells implies a failure of the APC presentation or recognition process.
Exogenously provided IL-2 can restore, at least in part, in an in vitro assay system with AIDS patients, cell mediated immunity activity to Human Immunodeficiency Virus (hereinafter "HIV"). This suggests that with AIDS there is also a deficiency in IL-2 production, even though IL-1 production is above normal. AIDS infected patients also have either poor or ineffective antibody dependent cellular cytotoxicity (hereinafter "ADCC"), antibody complement cytotoxicity (hereinafter "ACC") or natural killer (hereinafter "NK") activity, even at early stages before any clinical signs of AIDS related complex (hereinafter "ARC") or AIDS.
In an embodiment of the present invention, a vaccine for diseases, such as AIDS, is provided which specifically stimulates cellular immunity to diseases, such as AIDS.
There are currently a series of in vitro assays for cell mediated immunity which use cells from the host both as the substrate cell that initiates or stimulates the reaction for which the assay has been developed and as the target to assess cell mediated immunity. These in vitro assays include the cytotoxic T lymphocyte assay (hereinafter "CTL"); lymphoproliferative assays, e.g., tritiated thymidine incorporation; the protein kinase assays, the ion transport assay and the lymphocyte migration inhibition function assay (hereinafter "LIF") (Hickling, J. K. et al, J. Virol., 61:3463 (1987); Hengel, H. et al, J. Immunol., 139:4196 (1987); Thorley-Lawson, D. A. et al, Proc. Natl. Acad. Sci. USA, 84:5384 (1987); Kadival, G. J. et al, J. Immunol., 139:2447 (1987); Samuelson, L. E. et al, J. Immunol., 139:2708 (1987); Cason, J. et al, J. Immunol. Meth., 102:109 (1987); and Tsein, R. J. et al, Nature, 293:68 (1982)). These assays are disadvantageous in that they may lack true specificity for cell mediated immunity activity, they require antigen processing and presentation by an APC of the same MHC type, they are slow (sometimes lasting several days), and some are subjective and/or require the use of radioisotopes.
In an embodiment of the present invention, a diagnostic assay for cell mediated immunity is provided which overcomes the above-described problem.